Mycobacterium bovis strain bacillus Calmette-Guérin-induced liver granulomas contain a diverse TCR repertoire, but a monoclonal T cell population is sufficient for protective granuloma formation.

Granuloma formation is a form of delayed-type hypersensitivity requiring CD4(+) T cells. Granulomas control the growth and dissemination of pathogens, preventing host inflammation from harming surrounding tissues. Using a murine model of Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) infection we studied the extent of T cell heterogeneity present in liver granulomas. We demonstrate that the TCR repertoire of granuloma-infiltrating T cells is very diverse even at the single-granuloma level, suggesting that before granuloma closure, a large number of different T cells are recruited to the lesion. At the same time, the TCR repertoire is selected, because AND TCR transgenic T cells (Valpha11/Vbeta3 anti-pigeon cytochrome c) are preferentially excluded from granulomas of BCG-infected AND mice, and cells expressing secondary endemic Vbeta-chains are enriched among AND cells homing to granulomas. Next, we addressed whether TCR heterogeneity is required for effective granuloma formation. We infected 5CC7/recombinase-activating gene 2(-/-) mice with recombinant BCG that express pigeon cytochrome c peptide in a mycobacterial 19-kDa bacterial surface lipoprotein. A CD4(+) T cell with a single specificity in the absence of CD8(+) T cells is sufficient to form granulomas and adequately control bacteria. Our study shows that expanded monoclonal T cell populations can be protective in mycobacterial infection.

Mycobacterium bovis BCG-induced granuloma formation depends on gamma interferon and CD40 ligand but does not require CD28.

Progressive granuloma formation is a hallmark of chronic mycobacterial infection. Granulomas are localized, protective inflammatory reactions initiated by CD4+ T cells, which contribute to control of bacterial growth and blockade of bacterial dissemination. In order to understand the costimulatory requirements that allow CD4+ T cells to directly or indirectly induce granulomas, we studied granuloma formation after 6 weeks in Mycobacterium bovis BCG-infected CD28- and CD40 ligand (CD40L)-deficient mice and compared it to granuloma formation in infected wild-type inbred mice and infected cytokine-deficient mice. We characterized granulomas morphologically in liver sections, analyzed granuloma infiltrating cells by flow cytometry, and measured cytokine production by cultured granuloma cells. CD28-deficient mice have no defect at the local inflammatory site, inasmuch as they form protective granulomas and control bacterial growth. However, there are fewer activated T cells in the spleen compared to infected wild-type animals, and quantitative differences in the cellular composition of the granuloma are observed by flow cytometry. In CD40L-deficient mice, the granuloma phenotype is very similar to the phenotype in gamma interferon (IFN-gamma)-deficient mice. Both IFN-gamma-deficient and CD40L-deficient mice form granulomas which prevent bacterial dissemination, but control of bacterial growth is significantly impaired. The relative proportion of CD4+ T cells in granulomas from both CD28(-/-) and CD40L(-/-) mice is significantly decreased compared with wild-type animals. Both models demonstrate that the phenotype and activation stage of systemic T cells do not always correlate with the phenotype and activation stage of the localized granulomatous response.

T-Cell epitopes and human leukocyte antigen restriction elements of an immunodominant antigen of Blastomyces dermatitidis.

Humans infected with the dimorphic fungus Blastomyces dermatitidis develop strong T-lymphocyte responses to WI-1, an immunodominant antigen that has been shown to elicit protective immunity in mice. In the present study, the T-cell epitopes of WI-1 and human leukocyte antigen (HLA) restricting elements that display them were investigated. Peripheral blood mononuclear cells (PBMC) from 37 patients with a confirmed history of blastomycosis were tested for a response to WI-1 in primary proliferation assays; PBMC from 35 (95%) responded. Six patients whose PBMC proliferated strongly in response to WI-1 (defined as a stimulation index greater than 50) were tested further for responses to subcloned, recombinant fragments of the antigen. These patients responded chiefly to sequences within the N terminus and the 25-amino-acid tandem repeat. Cloned CD4(+) T cells from an infected individual were used to delineate more precisely the peptide epitopes in the fragments and HLA restricting elements that present them. A majority of the T-cell clones recognized an epitope spanning amino acids 149 to 172 within the N terminus, displayed by HLA-DR 15. A minority of the clones, which have been shown to perform a cytolytic function in vitro, recognized an epitope in the tandem repeat displayed by HLA-DPw4, an uncommon restricting element. Tandem repeat epitopes required display by the beta chain of DPw4 heterodimers. Thus, human T cells with different functions in vitro also recognize distinct regions of WI-1, raising the possibility that HLA restricting elements that present them could modulate immunity during blastomycosis by selection and display of WI-1 peptides.

TCR specificity in infection induced granulomas.

Granuloma formation is an essential host response to many intracellular pathogens and some particulate antigens. T lymphocytes, especially CD4+ T-cells, are required for the initial formation and ongoing maintenance of the inflammatory response. In the absence of CD4+ T-cells, most infections which normally provoke a granulomatous response are more widely disseminated or lethal since the protective lesions are either malformed or absent. The role of T-cell receptor mediated antigen specificity in infectious states is reviewed with a special emphasis upon recent work on S. mansoni induced granulomas.

African strains of Blastomyces dermatitidis that do not express surface adhesin WI-1.

African strains of Blastomyces dermatitidis differ from North American strains in their growth, morphology, and clinical disease phenotype. In addition, two serotypes, designated 1 and 2, have been described. We investigated African strains of B. dermatitidis for expression of the surface protein adhesin WI-1 and found that serotype 2 strains do not express it because they lack the coding sequence in their genome. The defect will make the strains useful for gene complementation and for testing the pathogenetic role of the WI-1 adhesin.

Transforming DNA integrates at multiple sites in the dimorphic fungal pathogen Blastomyces dermatitidis.

Blastomyces dermatitidis is a primary fungal pathogen of man and other mammals, but like many other human fungal pathogens, relatively little is known about the factors that account for its virulence and pathogenicity. We developed a transformation system to facilitate molecular genetic studies of putative virulence factors from B. dermatitidis. Transformation of the multinucleate yeasts was achieved by electroporation of DNAs containing a dominant selectable marker, hygromycin B (HygB) resistance. Southern analysis showed that transforming DNA invariably integrated ectopically into the chromosome. No evidence was found for extrachromosomal DNA. The HygB resistance could be expressed by either a 375-bp promoter fragment of the B. dermatitidis WI-1 gene encoding adhesin or an Aspergillus gpdA promoter placed 5' of the E. coli hph gene. Primer extension analysis showed that for plasmids containing the WI-1 promoter, transcription of the hph gene initiated within the 375-bp WI-1 promoter fragment. The combination of gene transfer and two promoters capable of independent transcription will allow us to restore or augment gene expression in appropriate strains and test an influence on virulence. Molecular genetic manipulation of B. dermatitidis represents a major advance in our ability to investigate the pathogenesis of blastomycosis and other similar fungal diseases.

Virulence factors of medically important fungi.

Human fungal pathogens have become an increasingly important medical problem with the explosion in the number of immunocompromised patients as a result of cancer, steroid therapy, chemotherapy, and AIDS. Additionally, the globalization of travel and expansion of humankind into previously undisturbed habitats have led to the reemergence of old fungi and new exposure to previously undescribed fungi. Until recently, relatively little was known about virulence factors for the medically important fungi. With the advent of molecular genetics, rapid progress has now been made in understanding the basis of pathogenicity for organisms such as Aspergillus species and Cryptococcus neoformans. The twin technologies of genetic transformation and "knockout" deletion construction allowed for genetic tests of virulence factors in these organisms. Such knowledge will prove invaluable for the rational design of antifungal therapies. Putative virulence factors and attributes are reviewed for Aspergillus species, C. neoformans, the dimorphic fungal pathogens, and others, with a focus upon a molecular genetic approach. Candida species are excluded from coverage, having been the subject of numerous recent reviews. This growing body of knowledge about fungal pathogens and their virulence factors will significantly aid efforts to treat the serious diseases they cause.

Genomic cloning, characterization, and functional analysis of the major surface adhesin WI-1 on Blastomyces dermatitidis yeasts.

WI-1 is a 120-kDa surface protein adhesin on Blastomyces dermatitidis yeasts that binds CD18 and CD14 receptors on human macrophages. We isolated and analyzed a clone of genomic WI-1 to characterize this key adherence mechanism of the yeast. The 9.3-kilobase insert contains an open reading frame of 3438 nucleotides and no introns. The amino acid sequence of native WI-1 matches the deduced sequence of genomic WI-1 at positions 757-769, 901-913, and 1119-1138, demonstrating the cloned gene is authentic WI-1. The complete coding sequence has 30 highly conserved repeats of 24 amino acids arrayed in tandem in two noncontiguous regions of the protein. The repeat sequence is homologous to the Yersiniae adhesin invasin, the C terminus displays an epidermal growth factor-like domain, and the N terminus has a short hydrophobic sequence that may be a membrane-spanning domain. The tandem repeats are predicted to be at the exposed surface of the protein, thereby explaining the adhesive properties of WI-1. The WI-1 promoter contains a CAAT box (nucleotide positions 2287-2290), TATA box (2380-2385), and CT motif (2399-2508). Transcription is initiated within the CT motif at nucleotide 2431. A 5.5-kilobase subclone containing the full coding sequence of WI-1 was expressed as a histidine-tagged fusion protein in Escherichia coli. Recombinant WI-1 has the expected molecular mass of 120 kDa, is strongly recognized in Western blots by rabbit anti-WI-1 antiserum, and binds human macrophage receptors in the same manner as native WI-1. This work clarifies a key adherence mechanism of B. dermatitidis and will permit further analysis of WI-1-mediated attachment to host cells, receptors, and extracellular matrix.

Altered expression of surface protein WI-1 in genetically related strains of Blastomyces dermatitidis that differ in virulence regulates recognition of yeasts by human macrophages.

The molecular basis for pathogenicity and virulence of the dimorphic fungus Blastomyces dermatitidis remains unknown. WI-1 is a major cell wall protein of B. dermatitidis yeasts and is a recognition target of both humoral and cell-mediated immunity. As an initial study to determine if WI-1 might be linked to virulence of B. dermatitidis, we quantified WI-1 expression on three genetically related strains that differ in their virulence for mice: wild-type virulent ATCC strain 26199, mutant ATCC strain 60915 (which is 10,000-fold reduced in virulence), and mutant ATCC strain 60916 (which is avirulent). Two principal alterations in WI-1 expression were observed in the mutants. First, the mutants express more WI-1 on their surface, as quantified by flow cytometry with monoclonal antibody to WI-1 and by radioimmunoassay, but the WI-1 on their cell wall is less extractable than that on the wild-type strain. Second, the mutants shed less WI-1 during culture and demonstrate impaired processing of shed WI-1. Surface alterations in WI-1 were accompanied by significant differences in the binding of the virulent and mutant strains to human monocyte-derived macrophages. Attachment of yeasts to macrophages paralleled and was proportional to the expression of WI-1. Compared with wild-type yeasts, both mutants bound to macrophages more rapidly and in two- to threefold-greater magnitude. Furthermore, about 75% of yeast binding to macrophages was inhibited by a Fab anti-WI-1 monoclonal antibody. These results suggest that altered WI-1 expression on attenuated and avirulent mutant B. dermatitidis yeasts greatly facilitates macrophage recognition and binding of yeasts and, in turn, may contribute to more rapid ingestion and killing in the host.

Altered expression of surface alpha-1,3-glucan in genetically related strains of Blastomyces dermatitidis that differ in virulence.

Recent studies of the dimorphic fungal pathogens Histoplasma capsulatum and Paracoccidioides brasiliensis have suggested a role in virulence for the cell surface carbohydrate alpha-(1,3)-glucan. To investigate a possible basis for alpha-(1,3)-glucan in the pathogenicity and virulence of the dimorphic fungus Blastomyces dermatitidis, we examined three genetically related strains of B. dermatitidis that differ in their virulence for mice: wild-type virulent strain ATCC 26199; mutant strain ATCC 60915, which is 10,000-fold reduced in virulence; and mutant strain ATCC 60916, which is avirulent. Immunologic quantitation of cell wall alpha-(1,3)-glucan revealed that the mutant yeasts were almost devoid of this sugar moiety, in contrast to the high concentration of alpha-(1,3)-glucan on the cell wall of the wild-type yeasts. These differences are discussed in relation to previous studies of yeast surface expression of the WI-1 antigen and recognition and binding of the related strains by human monocyte-derived macrophages.

Immunologic recognition of a 25-amino acid repeat arrayed in tandem on a major antigen of Blastomyces dermatitidis.

A 120-kD glycoprotein antigen abundantly expressed on Blastomyces dermatitidis yeasts is a target of cellular and humoral immune responses in human infection. To investigate the antigen and immune response more carefully at the molecular level, we screened an expression library from B. dermatitidis to identify clones that encode this antigen, designated WI-1. A 942-bp cDNA was isolated by immunologic screening with polyclonal, rabbit anti-WI-1 antiserum. Northern hybridization analysis showed that the cDNA hybridized to yeast message approximately equal to 3.9 kb. DNA and deduced protein sequence analysis of the clone demonstrated a 25-amino acid repeat arrayed in tandem, present in 4.5 copies near the 5' end, and rich in predicted antigenic epitopes. Further analysis showed strong homology in these tandem repeats with invasin, an adhesin of Yersiniae. Cloned cDNA was used to express a 30-kD fusion protein strongly recognized in western blots by rabbit anti-WI-1 antiserum, and by sera from all 35 blastomycosis patients studied. The fusion protein product of subcloned cDNA encoding only the tandem repeat also was strongly recognized in western blots by sera from the 35 blastomycosis patients, but not by sera from 10 histoplasmosis and 5 coccidioidomycosis patients. An antigen-inhibition radioimmunoassay showed that the tandem repeat alone completely eliminated rabbit and human anti-WI-1 antibody binding to radiolabeled native WI-1. From these results, we conclude that the 25-amino acid repeat of WI-1 displays an immunodominant B cell epitope, and that the carboxyl-terminus of the molecule exhibits an architecture that may promote adhesion of Blastomyces yeasts to host cells or extracellular matrix proteins and ultimately provide a clearer picture of the molecular pathogenesis of blastomycosis.